1. Field of the Invention
The present invention generally relates to a method for the cryopreservation of biological samples. More particularly, the present invention relates to a method for the cryopreservation of biological samples in solution based on very fast cooling rates achievable by contacting small droplets of the solution on a very cold solid surface. The cryopreservation method has been found to be particularly useful in the cryopreservation of oocytes and embryos, in particular mammalian embryos.
2. Background of Related Art
It is frequently the case that one desires to maintain biological samples for long periods of time, for example when the sample is desired to be used for a particular purpose at a later time. Unfortunately, as recognized by those of ordinary skill in the art, the freezing, cryopreservation and thawing, of biological samples using presently available techniques often is less than optimal, as biological activity of the biological sample is often significantly diminished. As the practical use of certain biological techniques dictates certain cells be stored for long periods of time, there has been a growing demand for new method of preserving biological samples, in particular cellular material, such the biological functionality of the material is preserved after warming. A particular need for the preservation of biological materials has arisen in germ plasma, in vitro embryo production and nuclear transfer.
There has been an increasing effort to store the oocytes and embryos of many animal species. These efforts have arisen from a number of human desires including, but not limited to, the desire to preserve animal species in numerical decline, to breed animals by in vitro fertilization techniques to increase genetic diversity and to overcome infertility problems, and to clone animals with high economic potential. With the growing interest in oocyte preservation, there has become an increasing awareness that current biological preservation techniques are less than adequate to preserve the oocytes of many animals species. For example, it is known that cattle oocytes are very sensitive to low temperatures. Despite the efforts of numerous research groups (See review by Palasz et al., Biotechnol. Adv. 14: 127-149 (1996)) cattle oocyte preservation remains a difficult task. Only a limited number of publications reported blastocyst and subsequent calf development from cryopreserved oocytes and the results remain inefficient (Fuku et al., Cryobiology 29: 485-492 (1992); Hamano et al., Theriogenology 38: 1085-1090 (1992); Otoi et al., Theriogenology 38: 711-719 (1992); Otoi et al., J. Reprod. Dev. 41: 361-366 (1995); Suzuki et al., Cryobiology 33: 515-524 (1996); Kubota et al., Mol. Reprod. Dev. 51: 281-286 (1998); Vajta et al., Mol. Reprod. Dev. 51: 53-58 (1998)).
It has been reported that the fertilization process can be compromised by the effect of cryoprotectants. A number of reports state that elevated concentrations of presently available cryoprotectants will have high toxicity with respect to oocytes and a negative effect on the subsequent development of oocytes and embryos (See, e.g., Martino et al., Mol. Reprod Der. 45: 503-512 (1996); Palasz et al., Biotechnology Advances 14: 127-149 (1996); Parks et al., Theriogenology 37: 59-73 (1992); Schellander et al., Theriogenology 42: 909-915 (1994); Vajta et al., Embryo Transfer Newsletter 15:12-18 (1997)). There are also reports of oocyte toxicity related to the cooling of cryopreservation (Parks et al., Theriogenology 37: 59-73 (1992)). For example, exposure to low temperature is known to result in de-polymerization of the meiotic spindle (Parks et al., Theriogenology 37: 59-73 (1992); Peura et al., Theriogenology 51: 211 (1999) (abstr.)). Previous experiments with frozen/thawed matured oocytes showed a significant reduction of enucleation rates when compared to fresh oocytes (Kubota et al., Mol. Reprod. Dev. 51: 281-286 (1998)).
Efforts today have centered on improving the survival rates of stored oocytes by improving cryopreservation techniques. According to Martino et al. (Martino et al., Biol. Reprod. 54: 1059-1069 (1996)), such efforts have focused on comparing different cryoprotectants (Otoi et al., Theriogenology 40: 801-807 (1993); Dinnyes et al., Cryobiology 31: 569-570 (1994)) and different freezing regimens (Lira et al., Theriogenology 35: 1225-1235 (1991)); or related vitrification methods (Otoi et al., Theriogenology 40: 801-807 (1993); Otoi et al., Cryobiology 37: 77-85 (1998)).
By “cryopreservation” it is meant the storage of biological materials at below the freezing point of water such that the material does not decompose. By “vitrification” it is meant a process of cooling biological material, employing cryoprotectants (chemicals that protect water from freezing) to inhibit the formation of ice in the cooling process, to a temperature about −100 degrees Celsius or lower, such that the solution containing the biological material reaches its glass transition temperature, that is the molecules cease to move relative to each other. It is recognized in the art that ice formation is damaging to biological material, in that it forces the material into shrinking pockets of residual unfrozen solution. As cooling continues, more than eighty percent of tissue volume can become converted to ice, and cells crushed beyond recovery. During vitrification, liquid water molecules maintain their natural random arrangements during deep cooling. There is no disturbance of other chemicals or cell components. Successful vitrification techniques make use of supercooling, that is cooling below the freezing point of the cryoprotection solution without freezing. Cryoprotectants are typically toxic to cells at high concentrations. Rapid freezing is believed to work by decreasing the concentration of cryoprotectant necessary to protect against ice crystal formation, thereby preserving the tissue at non-toxic concentrations of cryoprotectants.
It is believed that one of the bottlenecks of vitrification technology is the “insufficient” cooling rate of oocytes in current vitrification schemes (Vajta et al., Embryo Transfer Newsletter 15: 12-18 (1997)). In order to overcome this problem, several methods have been proposed which use very small amounts of solution
So-called “minimum drop vitrification” systems have allowed breakthrough results with bovine and porcine oocyte cryopreservation (See, e.g., Arav A., Vitrification of oocyte and embryos, In: Lauria A, Gandolfi F (eds.), New trends in embrvo transfer, Cambridge, England: Portland Press, 255-264 (1992)). In “minimum drop vitrification” small amounts of solution are placed on a special cryo-stage which is cooled down quickly. This method, unfortunately, has not been found by the art to be convenient for preserving large numbers of oocytes.
Other vitrification techniques have been reported. Methods utilizing few microliters of vitrification solution loaded into glass capillaries (Dinnyes et al., Cryobiology 31: 569-570 (1994)), or into open pulled plastic straws (Vajta et al., Mol. Reprod. Dev. 51: 53-58 (1998)) and then plunged quickly into liquid N2 were successfully tested for bovine oocyte vitrification. Similarly vitrification success was achieved by plunging oocyte-containing vitrification solutions with a small loop (Lane et al., Theriogenology 51: 167 (1999) (abstr.)). However, such techniques have not been found highly efficient presumably because plunging a warm object into liquid N2 results in the boiling of the liquid and for a short time creates an isolating layer of N2 vapor around the object.
In order to reduce the possibility of an isolating layer of vapor interfering with efficient vitrification, it has been proposed that oocyte-containing vitrification solution be dropped directly into liquid N2. Such technique has been reported to be more effective than prior art vitrification techniques. (Riha et al., Zivoc. Vir. 36: 113-120 (1991); Papis et al., Theriogenology 51: 173 (1999) (abstr.); Yang et al., Theriogenology 51: 178 (1999) (abstr.)), presumably by eliminating the insulation effect of the vapor. However, such technique suffers from the problem of vitrified oocyte retrieval.
Some groups have reported improved success of the cryopreservation of biological materials by using metal surfaces cooled down with the aid of liquid N2. Such metal surfaces are asserted to provide a more efficient heat transfer and to increase further the cooling rates than the cryo-stages used in minimum drop vitrification. Drosophila embryos were successfully preserved by placing them in a metal grid on a cold metal surface (Steponkus et al., Nature 345: 170-172 (1990)). Again, presently available techniques employing cooled metal surfaces have not been found convenient for preserving large numbers of oocytes.
Currently, as a consequence of the limited survival of preserved oocytes, and the cumbersome techniques available, in vitro embryo production and nuclear transfer experiments in cattle rely on freshly collected oocytes. This is a major limitation to numerous research teams and presents an obstacle to efficient planning and organizing of experiments.
There is a need therefore for improved biological material preservation methods, particularly those useful for preserving oocytes and other fragile cells. The method should permit the sterile handling of the biological material and preserve biological activity and viability for long periods of time.